Label-free three-dimensional imaging and quantitative analysis of living fibroblasts and myofibroblasts by holotomographic microscopy.

fibrosis holotomographic microscopy label‐free myofibroblasts quantitative phase imaging

Journal

Microscopy research and technique
ISSN: 1097-0029
Titre abrégé: Microsc Res Tech
Pays: United States
ID NLM: 9203012

Informations de publication

Date de publication:
10 Jul 2024
Historique:
revised: 18 06 2024
received: 20 05 2024
accepted: 24 06 2024
medline: 10 7 2024
pubmed: 10 7 2024
entrez: 10 7 2024
Statut: aheadofprint

Résumé

Holotomography (HT) is a cutting-edge fast live-cell quantitative label-free imaging technique. Based on the principle of quantitative phase imaging, it combines holography and tomography to record a three-dimensional map of the refractive index, used as intrinsic optical and quantitative imaging contrast parameter of biological samples, at a sub-micrometer spatial resolution. In this study HT has been employed for the first time to analyze the changes of fibroblasts differentiating towards myofibroblasts - recognized as the main cell player of fibrosis - when cultured in vitro with the pro-fibrotic factor, namely transforming growth factor-β1. In parallel, F-actin, vinculin, α-smooth muscle actin, phospho-myosin light chain 2, type-1 collagen, peroxisome proliferator-activated receptor-gamma coactivator-1α expression and mitochondria were evaluated by confocal laser scanning microscopy. Plasmamembrane passive properties and transient receptor potential canonical channels' currents were also recorded by whole-cell patch-clamp. The fluorescence images and electrophysiological results have been compared to the data obtained by HT and their congruence has been discussed. HT turned out to be a valid approach to morphologically distinguish fibroblasts from well differentiated myofibroblasts while obtaining objective measures concerning volume, surface area, projection area, surface index and dry mass (i.e., the mass of the non-aqueous content inside the cell including proteins and subcellular organelles) of the entire cell, nuclei and nucleoli with the major advantage to monitor outer and inner features in living cells in a non-invasive, rapid and label-free approach. HT might open up new research opportunities in the field of fibrotic diseases. RESEARCH HIGHLIGHTS: Holotomography (HT) is a label-free laser interferometric imaging technology exploiting the intrinsic optical property of cells namely refractive index (RI) to enable a direct imaging and analysis of whole cells or intracellular organelles. HT turned out a valid approach to distinguish morphological features of living unlabeled fibroblasts from differentiated myofibroblasts. HT provided quantitative information concerning volume, surface area, projection area, surface index and dry mass of the entire fibroblasts/myofibroblasts, nuclei and nucleoli.

Identifiants

pubmed: 38984377
doi: 10.1002/jemt.24648
doi:

Types de publication

Journal Article

Langues

eng

Sous-ensembles de citation

IM

Subventions

Organisme : MUR (Ministry of University and Research, Italy)
Organisme : Finanziamenti alla Ricerca di Ateneo
Organisme : University of Florence
Organisme : European Commission
Organisme : NextGenerationEU
Organisme : Missione 4 Componente 2
Organisme : Dalla ricerca all'impresa
Organisme : Innovation Ecosystem RAISE "Robotics and AI for Socio-economic Empowerment"
ID : ECS00000035

Informations de copyright

© 2024 Wiley Periodicals LLC.

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Auteurs

Francesca Sbrana (F)

Biophysics Institute, National Research Council, Genoa, Italy.

Flaminia Chellini (F)

Department of Experimental and Clinical Medicine, Section of Anatomy and Histology, Imaging Platform, University of Florence, Florence, Italy.

Alessia Tani (A)

Department of Experimental and Clinical Medicine, Section of Anatomy and Histology, Imaging Platform, University of Florence, Florence, Italy.

Martina Parigi (M)

Department of Experimental and Clinical Medicine, Section of Anatomy and Histology, Imaging Platform, University of Florence, Florence, Italy.

Rachele Garella (R)

Department of Experimental and Clinical Medicine, Section of Physiological Sciences, University of Florence, Florence, Italy.

Francesco Palmieri (F)

Department of Experimental and Clinical Medicine, Section of Physiological Sciences, University of Florence, Florence, Italy.

Sandra Zecchi-Orlandini (S)

Department of Experimental and Clinical Medicine, Section of Anatomy and Histology, Imaging Platform, University of Florence, Florence, Italy.

Roberta Squecco (R)

Department of Experimental and Clinical Medicine, Section of Physiological Sciences, University of Florence, Florence, Italy.

Chiara Sassoli (C)

Department of Experimental and Clinical Medicine, Section of Anatomy and Histology, Imaging Platform, University of Florence, Florence, Italy.

Classifications MeSH